Hello! My name is Mary, and I am a rising sophomore at San Francisco University High School. I am interested on the influence of culture on health care policies and plans to design an independent study to next year to research the subject. In my free time, I write and publish articles at a pharmaceutical start-up blog on alternative medicine and pharmaceuticals. I am a newbie when it comes to biotechnology so I am excited to learn more and practice some basic lab skills. Also, I am a contemporary play junkie and would be more than willing to go to a 2 hour play at 11 pm the day before a physics test.
Design Project: Glucose-6-phosphate dehydrogenase(G6PD)-generating Enteric Bacteria Glucose-6-phosphate dehydrogenase(G6PD) Deficiency is a hereditary condition in which an insufficient amount of G6PD is produced due to genetic mutation, and it affects more than 400 million people worldwide. G6PD is a rate-limiting enzyme that regulates many metabolic reactions in the body and is present in almost all cell types, although G6PD is expressed differently in each. G6PD is needed to maintain the normal function and lifespan of RBCs.
G6PD catalyzes the first key reaction in the Pentose Phosphate Cycle, the metabolic pathway of carbohydrates that oxidizes glucose-6-phosphate to generate NADPH2 and provide ribose-5-phosphate for nucleotide synthesis. Deficiency of G6PD occurs in all cells of the affected individual but is severe in RBCs because the pentose phosphate pathway is the only pathway to form NADPH2. NADPH2 maintains a reduced level of glutathione to maintain the normal structure of RBCs. Glutathione is an antioxidant that counteracts the toxicity of free radicals in the body. Without enough NADPH2, there are not enough glutathione to detoxify the harmful effects of free radicals, which leads to oxidative stress. Therefore, G6PD serves to maintain a healthy amount of RBCs in the body and protect RBCs from oxidative stress.
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Figure I- Pentose Phosphate Cycle This diagram shows that G6PD is needed in the first step to catalyze the reaction to form NADPH2.
G6PD Deficiency causes hemolysis, a condition in which RBCs die prematurely. Patients with severe G6PD Deficiency suffer from hemolytic anemia, which develops when RBCs die faster than they are produced, which leads to a decrease in oxygen for the body.
Figure II- RBCs affected by G6PD vs normal RBCs Oxidative stress denatures the hemoglobin of the RBCs causing them to form a “bite” shape.
The G6PD gene located on the telomeric region of the long arm of the X chromosome, which means that G6PD Deficiency is an X-linked recessive condition.[2] Because males only have one X chromosome, they only need one defective copy of the gene to be affected. On the other hand, females have two X chromosomes and would need two defective copies of the gene to be affected. Therefore, females are less likely than males to be affected by G6PD Deficiency. Figure IV- G6PD Gene This picture shows that G6PD is an X-linked recessive mutation. More males are affected than females.
The severity of G6PD Deficiency ranges among the affected population. Those who suffer from severe G6PD Deficiency experience chronic anemia and have almost no G6PD detected in their bodies. Those who suffer from moderate G6PD Deficiency have an unstable supply of G6PD but young RBCs are still healthy.
There are currently many different treatment options for G6PD Deficiency. Those with moderate G6PD Deficiency usually have a modified diet that does not include foods that could trigger oxidative stress, such as fava beans. Others take vitamins or other cofactors in the hopes that they will help the catalysis of NADPH2. Treatment for more severe cases of G6PD Deficiency include external enzyme replacement and gene therapy.
However, all of options above pose problems. Modified diet, vitamins, and detoxifying agents are only meant to relieve symptoms caused by G6DP Deficiency and therefore are only sometimes effective among those who have light or moderate G6PD Deficiency. These treatments are temporary relievers but they do not help with long-term relieve or solve the root problem. External enzyme replacement is impractical because it is very expensive. It costs over 140 U.S. dollars to buy 0.01 mg of 90% purified G6PD. In addition, the enzyme would have to be injected intravenously or subcutaneously, which could be inconvenient for many. Gene therapy would be the most effective treatment option but it currently faces many hurdles, such as the delivery of genes to the right cells and the control of expression of genes.
This project focuses on designing an internal enzyme replacement to treat G6PD deficiency. It allows bacteria (E. coli) to produce G6PD inside the small intestines.
Figure V- Pathway of G6PD Expression As shown, IPTG (inducer) activates the G6PD tac promoter, which then activates the G6PD gene. The G6PD gene produces G6PD which then catalyzes NADP into NADPH2 and Glucose-6-P into 6-Phosphogluconolactone through the PPC.
To enter the body, the bacteria (E coli.)would be placed inside a capsule and ingested into the body. The capsule would be dissolved and the bacteria travels to the small intestines and joins the flora of the small intestines.
The bacteria would then reproduce in the small intestines, which means that the supply of bacteria would not need to be replenished. This significantly cuts down the costs since it is not necessary to continuously purchase and ingest the bacteria. In addition, unlike the current treatment options, internal enzyme replacement aims to be a long-term treatment for G6PD Deficiency.
IPTG
G6PD
1
1
0
0
Figure VI- Truth Table If the system works, the truth table above applies. When the IPTC (inducer) is present, G6PD will be present. When the IPTC (inducer) is not present, G6PD will not be present.
One of the biggest challenge of this project is how the production of G6PD would be regulated. One possible solution is to add a constitutive promoter to the pathway, which would allow the bacteria to continuously produce small amounts of G6PD. If an excessive of amount of G6PD is produced, then the G6PD would supposedly be excreted. However, the ideal solution would be to indirectly link an inhibitor to the number of RBCs in the body. Another challenge is making sure that the G6PD produced by the bacteria is properly absorbed from the small intestines into the bloodstream.
To test if the system works, simply draw a blood sample to check the amount of RBCs in the blood and/or the level of G6PD in the blood. If the amount of RBCs in the blood and/or the level of G6PD in the blood is normal, then the system does work. If the amount of RBCs in the blood and/or the level of G6PD in the blood is below normal, then the system does not work.
Design Project: Glucose-6-phosphate dehydrogenase(G6PD)-generating Enteric Bacteria
Glucose-6-phosphate dehydrogenase(G6PD) Deficiency is a hereditary condition in which an insufficient amount of G6PD is produced due to genetic mutation, and it affects more than 400 million people worldwide. G6PD is a rate-limiting enzyme that regulates many metabolic reactions in the body and is present in almost all cell types, although G6PD is expressed differently in each. G6PD is needed to maintain the normal function and lifespan of RBCs.
G6PD catalyzes the first key reaction in the Pentose Phosphate Cycle, the metabolic pathway of carbohydrates that oxidizes glucose-6-phosphate to generate NADPH2 and provide ribose-5-phosphate for nucleotide synthesis. Deficiency of G6PD occurs in all cells of the affected individual but is severe in RBCs because the pentose phosphate pathway is the only pathway to form NADPH2. NADPH2 maintains a reduced level of glutathione to maintain the normal structure of RBCs. Glutathione is an antioxidant that counteracts the toxicity of free radicals in the body. Without enough NADPH2, there are not enough glutathione to detoxify the harmful effects of free radicals, which leads to oxidative stress. Therefore, G6PD serves to maintain a healthy amount of RBCs in the body and protect RBCs from oxidative stress.
Figure I- Pentose Phosphate Cycle
This diagram shows that G6PD is needed in the first step to catalyze the reaction to form NADPH2.
G6PD Deficiency causes hemolysis, a condition in which RBCs die prematurely. Patients with severe G6PD Deficiency suffer from hemolytic anemia, which develops when RBCs die faster than they are produced, which leads to a decrease in oxygen for the body.
Oxidative stress denatures the hemoglobin of the RBCs causing them to form a “bite” shape.
The G6PD gene located on the telomeric region of the long arm of the X chromosome, which means that G6PD Deficiency is an X-linked recessive condition.[2] Because males only have one X chromosome, they only need one defective copy of the gene to be affected. On the other hand, females have two X chromosomes and would need two defective copies of the gene to be affected. Therefore, females are less likely than males to be affected by G6PD Deficiency.
Figure IV- G6PD Gene
This picture shows that G6PD is an X-linked recessive mutation. More males are affected than females.
The severity of G6PD Deficiency ranges among the affected population. Those who suffer from severe G6PD Deficiency experience chronic anemia and have almost no G6PD detected in their bodies. Those who suffer from moderate G6PD Deficiency have an unstable supply of G6PD but young RBCs are still healthy.
There are currently many different treatment options for G6PD Deficiency. Those with moderate G6PD Deficiency usually have a modified diet that does not include foods that could trigger oxidative stress, such as fava beans. Others take vitamins or other cofactors in the hopes that they will help the catalysis of NADPH2. Treatment for more severe cases of G6PD Deficiency include external enzyme replacement and gene therapy.
However, all of options above pose problems. Modified diet, vitamins, and detoxifying agents are only meant to relieve symptoms caused by G6DP Deficiency and therefore are only sometimes effective among those who have light or moderate G6PD Deficiency.
These treatments are temporary relievers but they do not help with long-term relieve or solve the root problem. External enzyme replacement is impractical because it is very expensive. It costs over 140 U.S. dollars to buy 0.01 mg of 90% purified G6PD. In addition, the enzyme would have to be injected intravenously or subcutaneously, which could be inconvenient for many. Gene therapy would be the most effective treatment option but it currently faces many hurdles, such as the delivery of genes to the right cells and the control of expression of genes.
This project focuses on designing an internal enzyme replacement to treat G6PD deficiency. It allows bacteria (E. coli) to produce G6PD inside the small intestines.
Figure V- Pathway of G6PD Expression
As shown, IPTG (inducer) activates the G6PD tac promoter, which then activates the G6PD gene. The G6PD gene produces G6PD which then catalyzes NADP into NADPH2 and Glucose-6-P into 6-Phosphogluconolactone through the PPC.
To enter the body, the bacteria (E coli.)would be placed inside a capsule and ingested into the body. The capsule would be dissolved and the bacteria travels to the small intestines and joins the flora of the small intestines.
The bacteria would then reproduce in the small intestines, which means that the supply of bacteria would not need to be replenished. This significantly cuts down the costs since it is not necessary to continuously purchase and ingest the bacteria. In addition, unlike the current treatment options, internal enzyme replacement aims to be a long-term treatment for G6PD Deficiency.
Figure VI- Truth Table
If the system works, the truth table above applies. When the IPTC (inducer) is present, G6PD will be present. When the IPTC (inducer) is not present, G6PD will not be present.
One of the biggest challenge of this project is how the production of G6PD would be regulated. One possible solution is to add a constitutive promoter to the pathway, which would allow the bacteria to continuously produce small amounts of G6PD. If an excessive of amount of G6PD is produced, then the G6PD would supposedly be excreted. However, the ideal solution would be to indirectly link an inhibitor to the number of RBCs in the body. Another challenge is making sure that the G6PD produced by the bacteria is properly absorbed from the small intestines into the bloodstream.
To test if the system works, simply draw a blood sample to check the amount of RBCs in the blood and/or the level of G6PD in the blood. If the amount of RBCs in the blood and/or the level of G6PD in the blood is normal, then the system does work. If the amount of RBCs in the blood and/or the level of G6PD in the blood is below normal, then the system does not work.
https://docs.google.com/presentation/d/14ysROsuiXQiykRpHNiPln219TptjZGcOOlYD7e_PiP4/edit#slide=id.g1f970c1336_0_190